84 SAPONIFIABLE UPDDS 



ing off excess reagent, lipid spots appear brightly fluorescent in ultraviolet light. Free 

 fatty acids may be separated by reversed phase chromatography using paper impregnated 

 with rubber, silicones, paraffin oil, etc. General guides to paper chromatography give 

 much useful information on these techniques. There are also recent papers by Chayen 

 and Linday (36), and Ballance and Crombie (37). The latter authors were able to get 

 good separation of C10-C22 acids using paper impregnated with mineral oil and 70-95% 

 acetic acid as the solvent. Spots were revealed by converting the acids to insoluble cop- 

 per salts and then detecting copper with dithiooxamide. Sweeley and Moscatelli (38) have 

 developed a method for microanalysis of sphingolipids in which the long-chain bases 

 formed by hydrolysis are isolated and oxidized to aldehydes. The aldehydes can then be 

 characterized by gas chromatography. Gas chromatography has also been successfully 

 employed for the separation of fatty acids (or their methyl esters) (39). Thin layer chro- 

 matography of triglycerides and other fatty acid esters may replace paper chromatography 

 since it offers several advantages (40, 41). 



Spectral measurements have a special place in the characterization of saponifiable 

 lipids. Compounds with conjugated unsaturations are the only lipids to show absorption 

 peaks in the ultra-violet range from 210-400 m^. The only common fatty acid with con- 

 jugated double bonds is elaeostearic; and fats containing it or other conjugated trienes 

 show absorption at about 270 mji. Conjugated dienes absorb at about 230 m/i, tetraenes 

 at 310 mjLt, and pentaenes at 330 m/i. Conjugated systems containing acetylenic bonds 

 give about the same wavelength maxima as systems of ethylenic bonds, but they may be 

 recognized by other spectral differences such as intensity and side bands. The ultra- 

 violet spectrophotometry of fatty acids has been reviewed by Pitt and Morton (42). Ultra- 

 violet spectrophotometry may also be applied to non-conjugated unsaturated compounds by 

 first heating them 45 minutes at 180° C. in the presence of 7. 5% potassium hydroxide in 

 glycerol or ethylene glycol. This treatment isomerizes 1, 4 unsaturated systems to con- 

 jugated 1, 3 systems which then show the absorption spectra described above. By observ- 

 ing the spectrum before and after isomerization both conjugated and non-conjugated con- 

 stituents may be identified. This procedure can also be used for quantitative estimation 

 by weighing the sample of fat and measuring extinction coefficients at the appropriate 

 wavelengths for the different unsaturated acids. 



METABOLIC PATHWAYS 



The metabolic pathways for synthesis of the saponifiable lipids may be divided by 

 considering the biosynthesis of the products formed on hydrolysis and then the assembly 

 of these portions to make the complete lipid. Except for the fatty acids, biosynthesis of 

 other lipid components is covered in other chapters. Reviews on fatty acid biosynthesis 

 regularly appear in Annual Reviews of Biochemistry. Stumpf and Bradbeer (43) and Zill 

 and Cheniae (44) have reviewed fat metabolism in higher plants, and most of the pathways 

 summarized in Figures 5-1 and 5-2 will be found discussed in detail in their articles. 

 Biosynthesis of phosphatides and triglycerides is reviewed by Kennedy (45). 



In addition to the pathways shown in the diagram only a few points will be stressed 

 here. 



1. The conversion of fat to carbohydrate which is common in germinating seeds 

 probably occurs by way of the glyoxylate cycle described in Chapter 3. Fatty 

 acids are broken down to acetyl-CoA which enters the cycle and is converted 

 by way of malate and the malic enzyme to phosphoenolpyruvate. Phosphoenol- 

 pyruvate enters the reversible glycolytic sequence shown in Chapter 2. 



2. In fatty acid biosynthesis all intermediates are now regarded as bound to a multi- 

 functional enzyme through a thioester bond. Coenzyme A functions only in feed- 

 ing acetate and malonate to this system and in removing the final product as a 

 CoA derivative which can be utilized in the various ways shown on the diagram (44). 



